Polycaprolactone microspheres as carriers for dry powder inhalers : effect of surface coating on aerosolization of salbutamol sulfate

This study reports the factors controlling aerosolization of salbutamol sulfate (SS) from mixtures with polycaprolactone (PCL) microspheres fabricated using an emulsion technique with polyvinyl alcohol (PVA) as stabilizer. The fine particle fraction (FPF) of SS from PCL measured by a twin-stage impinger was unexpectedly found to be zero, although scanning electron microscopy showed that the drug coated the entire microsphere. Precoating the microspheres with magnesium stearate (MgSt) excipient solutions (1%–2%) significantly increased (p < 0.05, n = 5) the FPF of SS (11.4%–15.4%), whereas precoating with leucine had a similar effect (FPF = 11.3 ± 1.1%), but was independent of the solution concentration. The force of adhesion (by atomic force microscopy) between the PCL microspheres and SS was reduced from 301.4 ± 21.7 nN to 110.9 ± 30.5 nN and 121.8 ± 24.6 nN, (p < 0.05, n = 5) for 1% and 2% MgSt solutions, respectively, and to 148.1 ± 21.0 nN when coated with leucine. The presence of PVA on the PCL microspheres (detected by X-ray photoelectron spectroscopy) affected the detachment of SS due to strong adhesion between the two, presumably due to capillary forces acting between them. Precoating the microspheres with excipients increased the FPF significantly by reducing the drug–carrier adhesion. © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:733–745, 2012

Studies on the effect of the size of polycaprolactone microspheres for the dispersion of Salbutamol Sulfate from dry powder inhaler formulations

Purpose: To study the effect of the size of the surface-coated polycaprolactone (PCL) microparticle carriers on the aerosolization and dispersion of Salbutamol Sulfate (SS) from Dry Powder Inhaler (DPI) formulations. Methods: The microparticles were fabricated using an emulsion technique in four different sizes (25, 48, 104 and 150 μm) and later coated with Magnesium stearate (MgSt) and leucine. They were characterized by laser diffraction and SEM. The Fine Particle Fraction (FPF) of SS from powder mixtures was determined by a Twin Stage Impinger (TSI). Results: As the carrier size increased from 25 μm to 150 μm, the FPF of the SS delivered by the coated PCL particles increased approximately four fold. A linear relationship was found between the FPF and Volume mean Diameter (VMD) of the particles over this range. Conclusions: The dispersion behaviour of SS from PCL carriers was dependent on the inherent size of the carriers and the increased FPF of SS with increased carrier size probably reflects the higher mechanical forces produced due to the carrier-carrier collisions or collisions between the carrier particles and the internal walls of the inhaler during aerosolization.

Discovery and characterization of IGFBP-mediated endocytosis in the human retinal pigment epithelial cell line ARPE-19

Insulin-like growth factor binding proteins (IGFBPs) are prime regulators of IGF-action in numerous cell types including the retinal pigment epithelium (RPE). The RPE performs several functions essential for vision, including growth factor secretion and waste removal via a phagocytic process mediated in part by vitronectin (Vn). In the course of studying the effects of IGFBPs on IGF-mediated VEGF secretion and Vn-mediated phagocytosis in the RPE cell line ARPE-19, we have discovered that these cells avidly ingest synthetic microspheres (2.0 μm diameter) coated with IGFBPs. Given the novelty of this finding and the established role for endocytosis in mediating IGFBP actions in other cell types, we have explored the potential role of candidate cell surface receptors. Moreover, we have examined the role of key IGFBP structural motifs, by comparing responses to three members of the IGFBP family (IGFBP-3, IGFBP-4 and IGFBP-5) which display overlapping variations in primary structure and glycosylation status. Coating of microspheres (FluoSpheres®, sulfate modified polystyrene filled with a fluorophore) was conducted at 37 °C for 1 h using 20 μg/mL of test protein, followed by extensive washing. Binding of proteins was confirmed using a microBCA assay. The negative control consisted of microspheres treated with 0.1% bovine serum albumin (BSA), and all test samples were post-treated with BSA in an effort to coat any remaining free protein binding sites, which might otherwise encourage non-specific interactions with the cell surface. Serum-starved cultures of ARPE-19 cells were incubated with microspheres for 24 h, using a ratio of approximately 100 microspheres per cell. Uptake of microspheres was quantified using a fluorometer and was confirmed visually by confocal fluorescence microscopy. The ARPE-19 cells displayed little affinity for BSA-treated microspheres, but avidly ingested large quantities of those pre-treated with Vn (ANOVA; p < 0.001). Strong responses were also observed towards recombinant formulations of non-glycosylated IGFBP-3, glycosylated IGFBP-3 and glycosylated IGFBP-5 (all p < 0.001), while glycosylated IGFBP-4 induced a relatively minor response (p < 0.05). The response to IGFBP-3 was unaffected in the presence of excess soluble IGFBP-3, IGF-I or Vn. Likewise, soluble IGFBP-3 did not induce uptake of BSA-treated microspheres. Antibodies to either the transferrin receptor or type 1 IGF-receptor displayed slight inhibitory effects on responses to IGFBPs and Vn. Heparin abolished responses to Vn, IGFBP-5 and non-glycosylated IGFBP-3, but only partially inhibited the response to glycosylated IGFBP-3. Our results demonstrate for the first time IGFBP-mediated endocytosis in ARPE-19 cells and suggest roles for the IGFBP-heparin-binding domain and glycosylation status. These findings have important implications for understanding the mechanisms of IGFBP actions on the RPE, and in particular suggest a role for IGFBP-endocytosis.

Sulfate anion templation of macrocycles, capsules, interpenetrated and interlocked structures

This review examines the potential of anions, in particular sulfate, to template the formation of complex molecular architectures. Until recently, sulfate has been largely overlooked in this area and the examples described herein demonstrate this anion’s versatility in templating the formation of a diverse range of molecular systems including macrocycles, helixes, molecular capsules, interpenetrated and interlocked assemblies such as catenanes. In addition sulfate has been shown to template the formation of interpenetrated structures on a range of solid surfaces including gold, polystyrene beads and silicate nanoparticles, highlighting the potential of this anion in the fabrication of functional sensory devices exhibiting highly selective binding behaviour.

Near-infrared spectroscopic study of basic aluminum sulfate and nitrate

The tridecameric Al-polymer [AlO4Al12(OH)24(H2O)12]7+ was prepared by forced hydrolysis of Al3+ up to an OH/Al molar ratio of 2.2. Under slow evaporation crystals were formed of Al13-nitrate. Upon addition of sulfate the tridecamer crystallised as the monoclinic Al13-sulfate. These crystals have been studied using near-infrared spectroscopy and compared to Al2(SO4)3.16H2O. Although the near-infrared spectra of the Al13-sulfate and nitrate are very similar indicating similar crystal structures, there are minor differences related to the strength with which the crystal water molecules are bonded to the salt groups. The interaction between crystal water and nitrate is stronger than with the sulfate as reflected by the shift of the crystal water band positions from 6213, 4874 and 4553 cm–1 for the Al13 sulfate towards 5925, 4848 and 4532 cm–1 for the nitrate. A reversed shift from 5079 and 5037 cm–1 for the sulfate towards 5238 and 5040 cm–1 for the nitrate for the water molecules in the Al13 indicate that the nitrate-Al13 bond is weakened due to the influence of the crystal water on the nitrate. The Al-OH bond in the Al13 complex is not influenced by changing the salt group due to the shielding by the water molecules of the Al13 complex.

Synthesis of a heparan sulfate mimetic disaccharide with a conformationally locked residue from a common intermediate

A simple mimetic of a heparan sulfate disaccharide sequence that binds to the growth factors FGF-1 and FGF-2 was synthesized by coupling a 2-azido-2-deoxy-D-glucosyl trichloroacetimidate donor with a 1,6-anhydro-2-azido-2-deoxy--D-glucose acceptor. Both the donor and acceptor were obtained from a common intermediate readily obtained from D-glucal. Molecular docking calculations showed that the predicted locations of the disaccharide sulfo groups in the binding site of FGF-1 and FGF-2 are similar to the positions observed for co-crystallized heparin-derived oligosaccharides obtained from published crystal structures.

The PG500 series : novel heparan sulfate mimetics as potent angiogenesis and heparanase inhibitors for cancer therapy

Heparan sulfate mimetics, which we have called the PG500 series, have been developed to target the inhibition of both angiogenesis and heparanase activity. This series extends the technology underpinning PI-88, a mixture of highly sulfated oligosaccharides which reached Phase III clinical development for hepatocellular carcinoma. Advances in the chemistry of the PG500 series provide numerous advantages over PI-88. These new compounds are fully sulfated, single entity oligosaccharides attached to a lipophilic moiety, which have been optimized for drug development. The rational design of these compounds has led to vast improvements in potency compared to PI-88, based on in vitro angiogenesis assays and in vivo tumor models. Based on these and other data, PG545 has been selected as the lead clinical candidate for oncology and is currently undergoing formal preclinical development as a novel treatment for advanced cancer.

A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins

A surface plasmon resonance-based solution affinity assay is described for measuring the Kd of binding of heparin/heparan sulfate-binding proteins with a variety of ligands. The assay involves the passage of a pre-equilibrated solution of protein and ligand over a sensor chip onto which heparin has been immobilised. Heparin sensor chips prepared by four different methods, including biotin–streptavidin affinity capture and direct covalent attachment to the chip surface, were successfully used in the assay and gave similar Kd values. The assay is applicable to a wide variety of heparin/HS-binding proteins of diverse structure and function (e.g., FGF-1, FGF-2, VEGF, IL-8, MCP-2, ATIII, PF4) and to ligands of varying molecular weight and degree of sulfation (e.g., heparin, PI-88, sucrose octasulfate, naphthalene trisulfonate) and is thus well suited for the rapid screening of ligands in drug discovery applications.

Heparan sulfate mediates the proliferation and differentiation of rat mesenchymal stem cells

The growth and differentiation of mesenchymal stem cells is controlled by various growth factors, the activities of which can be modulated by heparan sulfates. We have previously underscored the necessity of sulfated glycosaminoglycans for the FGF-2-stimulated differentiation of osteoprogenitor cells. Here we show that exogenous application of heparan sulfate to cultures of primary rat MSCs stimulates their proliferation leading to increased expression of osteogenic markers and enhanced bone nodule formation. FGF-2 can also increase the proliferation and osteogenic differentiation of rMSCs when applied exogenously during their linear growth. However, as opposed to exogenous HS, the continuous use of FGF-2 during in vitro differentiation completely blocked rMSC mineralization. Furthermore, we show that the effects of both FGF-2 and HS are mediated through FGF receptor 1 (FGFR1) and that inhibition of signaling through this receptor arrests cell growth resulting in the cells being unable to reach the critical density necessary to induce differentiation. Interestingly, blocking FGFR1 signaling in post-confluent osteogenic cultures significantly increased calcium deposition. Taken together our data clearly suggests that FGFR1 signaling plays an important role during osteogenic differentiation, firstly by stimulating cell growth that is closely followed by an inhibitory affect once the cells have reached confluence. It also underlines the importance of HS as a co-receptor for the signaling of endogenous FGF-2 and suggests that purified glycosaminoglycans may be attractive alternatives to growth factors for improved ex vivo growth and differentiation of MSCs.

Bioactive mesopore-glass microspheres with controllable protein-delivery properties by biomimetic surface modification

Microsphere systems with the ideal properties for bone regeneration need to be bioactive, and at the same time possess the capacity for controlled protein/drug-delivery; however, the current crop of microsphere system fails to fulfill these properties. The aim of this study was to develop a novel protein-delivery system of bioactive mesoporous glass (MBG) microspheres by a biomimetic method through controlling the density of apatite on the surface of microspheres, for potential bone tissue regeneration. MBG microspheres were prepared by using the method of alginate cross-linking with Ca2+ ions. The cellular bioactivity of MBG microspheres was evaluated by investigating the proliferation and attachment of bone marrow stromal cell (BMSC). The loading efficiency and release kinetics of bovine serum albumin (BSA) on MBG microspheres were investigated after coprecipitating with biomimetic apatite in simulated body fluids (SBF). The results showed that MBG microspheres supported BMSC attachment and the Si containing ionic products from MBG microspheres stimulated BMSCs proliferation. The density of apatite on MBG microspheres increased with the length of soaking time in SBF. BSA-loading efficiency of MBG was significantly enhanced by co-precipitating with apatite. Furthermore, the loading efficiency and release kinetics of BSA could be controlled by controlling the density of apatite formed on MBG microspheres. Our results suggest that MBG microspheres are a promising protein-delivery system as a filling material for bone defect healing and regeneration.

Porous PLGA microspheres effectively loaded with BSA protein by electrospraying combined with phase separation in liquid nitrogen

Polymer microspheres loaded with bioactive particles, biomolecules, proteins, and/or growth factors play important roles in tissue engineering, drug delivery, and cell therapy. The conventional double emulsion method and a new method of electrospraying into liquid nitrogen were used to prepare bovine serum albumin (BAS)-loaded poly(lactic-co-glycolic acid) (PLGA) porous microspheres. The particle size, the surface morphology and the internal porous structure of the microspheres were observed using scanning electron microscopy (SEM). The loading efficiency, the encapsulation efficiency, and the release profile of the BSA-loaded PLGA microspheres were measured and studied. It was shown that the microspheres from double emulsion had smaller particle sizes (3-50 m), a less porous structure, a poor loading efficiency (5.2 %), and a poor encapsulation efficiency (43.5%). However, the microspheres from the electrospraying into liquid nitrogen had larger particle sizes (400-600 m), a highly porous structure, a high loading efficiency (12.2%), and a high encapsulation efficiency (93.8%). Thus the combination of electrospraying with freezing in liquid nitrogen and subsequent freeze drying represented a suitable way to produce polymer microspheres for effective loading and sustained release of proteins.

Bioactive inorganic materials/alginate composite microspheres with controllable drug-delivery ability

Alginate microspheres are considered a promising material as a drug carrier in bone repair due to excellent biocompatibility, but their main disadvantage is low drug entrapment efficiency and non-controllable release. The aim of this study was to investigate the effect of incorporating mesoporous bioglass (MBG), non-mesoporous bioglass (BG) or hydroxyapatite (HAp) into alginate microspheres on their drug-loading and release properties. X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and atomic emission spectroscopy (AES) were used to analyse the composition, structure and dissolution of bioactive inorganic materials and their microspheres. Dexamethasone (DEX)-loading and release ability of four microspheres were tested in phosphate buffered saline with varying pHs. Results showed that the drug-loading capacity was enhanced with the incorporation of bioactive inorganic materials into alginate microspheres. The MBG/Alginate microspheres had the highest drug loading ability. DEX release from alginate microspheres correlated to the dissolution of MBG, BG and HAp in PBS, and that the pH was an efficient factor in controlling the DEX release; a high pH resulted in greater DEX release, whereas a low pH delayed DEX release. In addition, MBG/alginate, BG/alginate and HAp/alginate microspheres had varying apatite-formation and dissolution abilities, which indicate that the composites would behave differently with respect to bioactivity. The study suggests that microspheres made of a composite of bioactive inorganic materials and alginate have a bioactivity and degradation profile which greatly improves their drug delivery capacity, thus enhancing their potential applications as bioactive filler materials for bone tissue regeneration.